Protein folding is the process by which provides proteins with their three-dimensional structure.
In order to be able to perform their specific biological assignment proteins have to be folded into a three-dimensional structure. This structure though is not stable in many cases, proteins can in parts or as a whole unfold and thus be in part or as a whole become denatured. When misfolding, a protein will form a structure which does not correspond to its native state. The disposition of partly or complete unfolding or alternative folding of its structure is a genuine feature of all proteins.
Whereas the mechanisms of checking such intracellular misfolded proteins/peptides—especially after polypeptide-synthesis and with mutations—have been widely known, extracellular misfolded proteins and their function have just recently become the subject of research.
Cells are very good at quality controlling as to protein foldings and usually correct or recycle misfolded intracellular proteins through chaperones, ubiquitin proteasome system.
The release buttons for misfoldings with extracellular proteins can vary: e.g., contact of proteins/peptides with glass, lipids, cellular membranes1, metals and metallic compounds and glucoses-polymere can cause such misfoldings2. Even pharmaceutical medication can be misfolded3.
The phenomenon of misfolded proteins is of particular importance for the investigation of surfacial biocompatibility of graft blood vessels such as cardiac supportive systems and cardiac valve replacement. But protein/peptide misfolding can also be induced by post-translational protein-modification such as glycation4,5, inevitably effecting “adevanced glycation” end-products or through modification with ROS6, HOCl, aldehydes—such as acrolein (propenal), 4-hydroxy-2-nonenal—and malondyaldehyd7, the impact of peptidyl-prolyl-cis/trans-isomerases and proteindisulfidisomerasis8, enzymatic splitting9, and through changes by glycolisation or glycolidases. The composition of the various peripherial conditions, detergents, the distribution of zinc10, Cu, Pb, or ethanol, pH-number, the bio-molecular concentration, contact to membranes and also cholesterol and sphingolipid concentration within the membrane11,12, and furthermore pressure, shear force and changes in temperature, even contact to other biomolecules have an impact on the misfolding of proteins. Microorganisms aimfully produce misfolded proteins in order to scotch-tape themselves to cellular hosts13. That is the way misfolded proteins take in the case of bacteriogenic, viral and mycotic infections, to enter into the blood circulatory system of their host. This same-self phenomenon might also play its part in the incidence of sepsis. Interesting enough, mammals, too, seem to make use of the misfolded proteins/peptides: the alpha-defensins of human granulocytics show effects like misfolded proteins in more than one way. They embed in contact to membranes, sequester the general bonding protein for misfolded proteins t-PA and bond—as we have recently succeeded in demonstrating—to the chaperone GRP78/BIP and furthermore they can activate thrombocytes14.
In 1968 Levinthal15 pointed out that the correct folding of a newly synthetised polypeptide would take a longer period of time than the lifespan of the universe itself, if all the possible conformations were tried out having been randomly searched for. As protein folding, however, will take just seconds or even milliseconds we assume that there are resilient interstages. The natural conformation of a protein is similar to the stage of the lowest possible (Gibbs) Enthalpy16. Minute variations as to surrounding elements can—as mentioned above—induce the partial generation of oligopeptides/polypeptides/proteins so that the latter again will take to resilient interstages. Partly unfolded/misfolded proteins show a certain disposition to associate with their likes which may induce oligomeric processes, aggregation and the forming of fibrils.
In the 1980s scientists found enzymes which managed to identify misfolded proteins and to cover up their hydrophobical surfaces, the so-called chaperones. To a limited extent these also have the ability to repair misfoldings by means of additional energy. This procedure may, though, induce new problems and the number of disorders based on misfolding proteins/peptides has been on the increase lately. This shows how important the correct protein folding is, and that the protein misfolding is a key mechanism to diseases, which result in serious impairment and disabilities. The misfolding of proteins has been found particularly with patients who suffer from neurodegenerative diseases such as the Alzheimer Disease, BSE, CJD (Creutzfeldt-Jakob-Disease), ALS (amyotrophic lateral sclerosis) or the Parkinson Disease17.
Misfoldings of proteins and protein-aggregates has furthermore been regarded lately as a mechanism inducing other serious health problems such as arteriosclerosis—inducing peripheral vascular obliteration, myocardial infarction and apoplexy, amyloidosis in context with dialysis, preeclampsy—a hypertensive disease during pregnancy18, and the immunogenicity of protein/peptide-holding pharmaceuticals19.
With a large number of misfoldings which could be identified as causes of disease, proteins are converted into orderly aggregates and fibrillars (amyloids) which escape cellular quality control and protein degradation. We do know, though, that in particular the low-molecular intermediates, oligomeres and aggregates are to be seen the causes of serious diseases.
The risk of falling ill through misfoldings of proteins/peptides increases alongside with aging. Scientific researchers assume that the quality control system for protein foldings decreases by aging and that thus the number of illnesses entailed increases20. The knowledge we have today on such matters supports the assumption that all misfolded proteins share a common structural mechanism which in most cases is supposed to cause cytoxicity. So oligomeres from misfolded proteins with cellular membranes can have double-bind effects and build up structures which will destroy the selective ionic permeability, and this may induce the perishing of the cells. Misfolded proteins, however, also have an impact directly on fibrinolysis and the kallikrein system, as a fibronectins-type-1 domain in the serine proteases tissue-plasminogen—activator (tPa) and FXII immediately identifies such proteins21,22.
The activation of fibrinolysis and of the contact-activating system could be seen as a mechanism to cut down or destroy dangerous extracellular misfolded proteins/peptides before they by means of fibrillas become more resistant against proteases23,24. In recent years chaperones like GRP78, also known as BIP, and others have been discovered on cellular surfaces. Whereas in the beginning GRP78 was assumed to be found on tumour cells exclusively, we have learned by now that GRP78 can also be detected on other cells under stress. Autoantibodies versus cell-membrane-hosted GRP78 are to be found with many tumour-affected patients particularly suffering from prostate adenocarcinoma, ovary or stomach cancer25. Subject to stress like hypoxia, shortage in or abundance of glucosis and the effects of shear force we find GRP78 for instance on endothelium cells, cardiomyocytes, monocytes/foam cells and muscle cells. GRP78 is mainly to be detected with advanced arteriosclerotic lesions and on the surface of the fibroid cap in apolipoprotein-deficient mice and with humans. GRP78 is particularly to be discovered at vascular sites which are rheologically particularly prone to arteriosclerotic disorders26-28. Chen et al. found out that statins (HMG-CoA) increase the expression of GRP7829.
Excessive expression of GRP78 inhibits the proagulante activities of Tissue Factor30. GRP78 may e seen as the regulating agent of the Tissue Factor Dependant coagulation31. The two independently working teams of Lina Badimon and Beate Kehrel also detected GRP78 also on the plasma-membrane of thrombocytes32,33. Herczenik and others have succeeded in presenting evidence that thrombocytes have been activated by misfolded proteins34. The team of Beate Kehrel was able to demonstrate that the activating of thrombocytes has been initiated by the various misfolded proteins such as HOCl-modified albumin, EAP from S. aureus35, an alpha-defensin on human neutrphil granulocytes36 and amyloid renal atrophic TSP-1 peptide RFYVVMWK due to GRP78. GRP78 thus seems to be a receptor on thrombocytes for alterated structures/misfoldings in proteins/peptides. The task of GRP78 depends on ATP37. Deng et al. have succeeded in showing that aspirin inhibits the activity of GRP78, in fibroblasts by inhibiting its ATP-activities38. Scientists of OxProtect GmbH have succeeded in presenting that aspirin/ASS by inhibiting the ATPase-activity of GRP78 also inhibits the activation of platelets by misfolded proteins. Therefore misfolded proteins can be specifically administered for monitoring the reaction of patients to a therapy with ASS/aspirin HOCl-modified misfolded proteins furthermore interact directly with the active metabolites of the thienopyridines. There are two assumptions of why HOCl-modified proteins can have a negative impact on the ADP-induced activating of thrombocytes. The one lies in the fact that HOCl-modified proteins actually can catch active metabolites of the thienopyridines so that less active metabolite is left for inhibiting the ADP-receptors P2Y12. Immobilised HOCl-modified proteins have actually been found in arteriosclerotic walls of blood vessels. Sugyama et al. have discovered HOCl-modified proteins directly under the thrombus at the plaque-erosion site when they examined the arteriosclerotic plaques which had caused the lethal stroke. Scientists of OxProtect GmbH have succeeded in identifying reactive groups on HOCl-modified proteins which bind with high affinity to free thiol groups. These react to the free thiol groups of the active metabolites of the thienopyridines like Clopidogrel and Prasugrel. As the thiol groups of active metabolites of the thienopyridine are essential to inhibiting for inhibiting P2Y12, there are fewer free metabolites for inhibiting P2Y12 in lower concentration. The other assumption is likely to refer to the fact that especially female patients who run a high risk of suffering from vascular incidents one day (myocardial infarction, stroke, peripheral vascular obliteration) have a higher concentration of HOCl-oxidated proteins in their blood; for these patients have been found to have higher concentrations of free myeloperoxidasis in plasma or serum, which is the one enzyme which is held to be responsible for the production of HOCl—as has been recently shown40-52.
As HOCl-modified proteins in their function of misfolded proteins are a major thrombocyte-activating factor—as shown by means of EP 1 328 289—and as on the other hand misfolded proteins can react to GRP78 on the surface of arteriosclerotic plaques, they themselves are very likely to be envolved in the process of the diseases and that at least some part of the protective mechanisms of the thienopyridines and ASS/Aspirin is based on the inhibiting of the pathogen effects of misfolded proteins.
As to the invention presented here the inventors have furthermore succeeded in discovering that HOCl-modified proteins can be used within laboratory methods for monitoring a thienopyride-based therapy.
One receptor for misfolded proteins on endothelial cells, even muscular cells and monocytes/makrophages is that GRP78. Successful bindings of polypeptides such as Kringel5 from plasminogen or ADAM15 are decisive for apoptose or proliferation, for life or death of the endothelial cell concerned53-55. On makrophages GRP78 is associated with G alpha q11. Such activated alpha 2 macroglobulins release a signalling chain in makrophages and tumour cells via GRP7856. Autoantibodies binding GRP78 on monocytes induce the production of TNF alpha57.
Misfolded proteins activate fibrinolysis. This may—as the case of endostatin has proved—lead to the detatching of the endothelial cells from the subendothelial matrix.58.
It seems highly probable that the cellular functions of GRP78 are under the influence of its misfolded protein ligands and therewith the presence and the degree of concentration of misfolded proteins plays a vital part in such essential issues as cell proliferation or apoptosis, wound healing and the generation of thrombosis.
GRP78 is involved in the cellular internalization of micro-organisms and proteins. Even peptides bound to GRP78 on cellular surfaces can get internalised59.
This might induce stress in the reticuloendothelial system. ER-stress has recently been identified as a prime mover for arteriosclerosis, type-II diabetis, obesity and their secondary diseases60-63. Life without any GRP78 is not possible. GRP78+/− heterocygous mice are resistant against diet-induced hyperinsulinemia, the generating of a steatohepatitis, inflammation within white adipose tissue and hyperglycemia. There is the interesting fact that these mice in spite of high-calorie, fatty diets did not put on weight64,65. In white adipose tissue GRP78 heterocygosity related to fatty diets induced an increase in adaptive response towards unfolded proteins (unfolded protein response (UPR)) and improved quality control of the endoplasmatic reticulum. That way obese and type II diabetic Grp78+/− mice recovered. These results clearly show the importance of quality control versus misfolded proteins for the homeostasis of energy-balance and glucosis metabolism.
ER-stress may induce apoptosis of even muscular cells. Thus ER-stress is very likely to contribute a fair share in plaque-rupturing66. And it is also likely that by generating a ligand thereof. OxLDL being a misfolded protein, too, modulates or even regulates the generating of foam cells which then is one of the initial steps toward arteriosclerosis.
In addition to cellular chaperones there are extracellular soluble proteins which may carry out chaperone-like tasks67. Among these proteins we find e.g., the alpha2 macroglobulins, clusterin (apolipoprotein 3), serum amyloid P and haptoglobins68-70. These proteins bind to misfolded proteins and facilitate the intracellular absorption of those complexes through Scavanger receptors such as “low-density lipoprotein receptor related to protein” (LRP, CD68, CD91), CD36, Scavanger receptor A, Scavanger receptor B-I, and RAGE. Complexes on misfolded proteins and chaperone-like soluble proteins can be detected in blood, plasma, and serum.
But also apolipo-proteins in particular apolipo-protein E (apoE), complement factors such as C1q and heparan-sulfate-proteoglycans can sequester misfolded proteins and thus contribute to their displaying their malicious effects on living organism. This has at length been described for amyloid beta, the culprit agent of the Alzheimer Disease71-73. Isoforms and variations among species have an impact on the effect of ApoE as to the self-association/aggregation and removal of amyloid beta protein.
Complexes of apolipoproteins or complement factors and misfolded proteins might thus become useful markers for disorders where misfolded proteins are involved, and heparan-sulfate-proteoglycans should contribute to the identification of misfolded proteins. Whether ApoE contributes via reaction to extracellular misfolded proteins towards the generating of arteriosclerosis is at present still a highly speculative matter.
Arteriosclerosis and resulting vascular diseases such as myocardial infarction, stroke, periphere vascular obliteration and the Alzheimer disease are related diseases. They both are based on a reaction of inflammation, on the impact of cholesterol, and sphingolipids in the cellular membrane and on the symptomatical appearance of misfolded proteins74.
In context with arteriosclerosis and diseases resulting thereof such as angina pectoris, myocardial infarction, TIA, stroke, peripheral vascular obliteration particularly the misfoldings of those proteins are of interest which have been induced by reaction to a product of myeloperoxidase or by sPLa2 and of particular interest on the grounds that these two enzymes themselves are excellent bio-markers of increased vascular risk75-77. Inhibitors of sPLA2 as therapeutical devices have already been put on clinical tests. Detection of sPLA2-activity induced misfoldings of LDL would therefore be highly desirable also for the monitoring of therapy with PLA-2 inhibitors. Amyloid insulin-residue have already been discovered with type-2 diabetics and other elderly patients.
Hyperinsulinemia- and hyperglycemia-induced resistance against insulin cause an increase in generating of plaque as to neuritis with Alzheimer Disease patients78,79. A dangerous feature of insulin is the disposition it shows of generating amyloid fibrillars. Misfolded insulin in the form of fibrillar insulin residue has been discovered with diabetic patients80,81. Insoluble insulin fibrillars may lead to a blockage of hypodermic needles for injections in the attempt of applying pharmaceutical insulin medication and are also held responsible for immunologic intolerance reactions which at times occur during therapy with pharmaceutical insulin medication82,83. Preliminary stages of fibrillars from misfolded proteins serve as a sort of solidification nucleus for the generation of larger aggregates and fibrillars. In addition to residue with type-II diabetics the generation of fibrillars forms, however, a big problem in production, storage, and application of soluble insulin in diabetic medication. Preliminary stages of fibrillars through misfolded proteins serve as a solidification nucleus for the generation of larger aggregates and fibrillars. Thus it is essential for the insulin production and for the application to the patient, to identify even partly unfolded/misfolded insulin and to eliminate this from medication and its preliminary stages as well. WO/2004/013176 describes such a method of cleansing preproinsulin which considerably reduces the forming of insulin fibrillars.
As misfoldings of extracellular proteins is such an essential phenomenon in the pathogenesis of diseases but misfolded proteins are also very useful as nano-materials, methods of detection of misfolded proteins/peptides have been described.
Misfolded proteins show some disposition for associating within their own kind84. In this procedure they can form oligomers, amorphous aggregates of various size or regularly ordered fibrillars. Such fabrillars are called amyloids. Some authors have already described misfolded proteins/peptimonomers, -oligomers and -aggregates as amyloids. In-vitro-generated fibrillars from misfolded proteins are also called amyloids by some other authors85. Fibrillars usually are of ca. 10 nm in diameter and can be of various lengths. Extracellular amyloid residue as to be found with the ordinary amyloidosis can be identified by the color of congo red when exposed to polarised light which then leads to a greenish double-refraction (“apple-green bi-refringence” so-called dichroism)86,87. Other methods of detection for discovering amyloid fibrillas are thioflavin S-fluorescence or the analysis of fibrillars by means of x-ray-diffraction. Both methods, though, are poor at detecting misfolded protein/peptide-monomers, -oligomers and aggregates or altogether fail in that respect.
In the beginning just filibrary structures were recognised in their pathogenic function, which form the basis of classic amyloidosis88,89—such as immunoglobulin-light chains90, amyloid A91, transthyretin92, cystatin C, apolipoprotein A-I93, gelsolin, fibrinogen Aalpha-chain, lysozyme, apolipoprotein A-II (2001), Islet amyloid polypeptidide (IAPP)94,95, leucocytes, chemotactic factor 2, LECT296, Alzheimer amyloid beta-peptides97, prion protein98-100, beta2 microglobulin101.
But now, after discovering that the misfoldings of proteins is not limited to just a few proteins but that it is a general feature it is absolutely essential to identify non-fibrillar conformations of misfolded proteins as well; and we have determined to find out about mechanisms which may be useful for all misfolded proteins/peptides or at least for a large part of these proteins/peptides for purposes of identification, sequestering, concentration and elimination.
Misfolded proteins according to the invention are very heterogeneous. They include monomer misfolded proteins/peptides, the range of small to large oligomers of misfolded proteins/peptides, large amorphous-looking aggregates of misfolded proteins/peptides up to large well-ordered fibrillar structures. They have in common—no matter what their amino acid sequence is—certain qualities which are very helpful in scientific research, medicine or for commercial use.
EP 1 380 290 describes the bond of misfolded proteins to the tissue-plasminogen-activator (tPa), the activating of the fibrinolysis by misfolded proteins and the use of fibrinolysis for fighting misfolded proteins.
EP 2 007 800 describes methods of identifying beta-structured misfolded proteins by bondings to chaperones, in particular to GRP78/BIP, clusterin, HSP72 or hapoglobins. In this respect others have been already mentioned as possible bonding proteins such as HSP60, HSP90, DNAK, HGFA, tPA, plasmogen, factor XII, IVIg, and the cellular receptors low density lipoprotein receptor related protein (LRP, CD91), CD36, Scavenger receptor Am, Scavenger receptor B-I, and RAGE.
EP 1 820 806 gives a description of an antibody which is able to identify misfolded proteins.
EP 2 058 000 describes the intensification of immune response by misfolded proteins and the use of misfolded proteins to support an immune response.
WO2007/008073 gives an insight into the impact of detecting and quantifying by comparing the contents of misfolded proteins in a sample with that in a corresponding sample after the test sample has been altered in a way which makes the alteration in the contents of misfolded proteins foreseeable. The method of identifying then is described as the activating of fibrinolysis via tPA and the activating of the kallikrein systems via FXIIa which in turn shares homologuous elements with tPA. That survey describes further binding substances for misfolded proteins such as thioflavin T (ThT), congo red, ThT, recombinant finger domains of tPA, FXII, HGFA, and fibronectins; serum amyloid P component (SAP), antibody versus misfolded proteins and a soluble fragment of the receptor “advanced glycation end-products” (sRAGE).